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Methods and Compositions for Direct Reprogramming of Somatic Cells to Stem Cells, and Uses of these Cells

a technology of somatic cells and compositions, applied in the field of compositions for direct reprogramming of somatic cells to stem cells, can solve the problems of major hurdles to overcome before induced cell technology, other challenges of cells, genetic instability and cancer risk,

Inactive Publication Date: 2014-09-18
THE RES FOUND OF STATE UNIV OF NEW YORK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes methods for generating induced stem cells (iSCs) from somatic cells by contacting them with an induction factor that reprograms them. The induction factor can be a genetic construct or a fusion protein. The somatic cells can be taken from various sources like amniotic fluid, bone marrow, blood, etc. The iSCs can be used for tissue repair and regeneration purposes like treating heart disease, stroke, diabetes, cancer, etc. The patent also provides a way to induce different types of iSCs like neural stem cells, endothelial stem cells, etc.

Problems solved by technology

However, in addition to the ethical and supply issues surrounding the use of human fetal tissue as a source of ES cells, ES cells have other challenges, including genetic instability and cancer risk.
However, while experiments with stem cell technologies show great promise, major hurdles remain to be overcome before induced cell technology can be considered safe for human treatment.
iPS cells, like embryonic stem (ES) cells, have numerous challenges, including genetic instability and cancer risk.
In addition, lentiviral or retroviral delivery could possibly cause a random insertion of the inducing gene into the genome and it is feasible that this delivery could happen within the coding sequence of a vital gene, thus disrupting the gene and causing a damaging mutation leading to developmental or malignant disorders.
The mature cells derived from direct reprogramming are likely insufficient for cellular therapy due to their limited capacity to self-renew and regenerate.
Recently, several published research accounts have reported the direct reprogramming of somatic skin cells to NSCs using a lentiviral vector expressing SOX2 or a combination of defined transcriptional factors However, the described process requires co-culturing the somatic cells with feeder cells, which carries additional risks.
Finally, the efficiency of direct reprogramming of such cells is very low, resulting in insufficient numbers for clinical use.
The safety issues and low efficiency of direct reprogramming are barriers for clinical applications of these cells.
Thus, there remain numerous barriers to be solved before these promising therapies are ready for use in human subjects.

Method used

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  • Methods and Compositions for Direct Reprogramming of Somatic Cells to Stem Cells, and Uses of these Cells
  • Methods and Compositions for Direct Reprogramming of Somatic Cells to Stem Cells, and Uses of these Cells
  • Methods and Compositions for Direct Reprogramming of Somatic Cells to Stem Cells, and Uses of these Cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

OCT4 or SOX2 Overexpression Via Lentiviral Expression De-Differentiates AF Cells

[0125]AF cells could be easily cultured and expanded in large amount from only 5 ml AF sample. The average doubling time of these cells is 12±1.6 hours. Passage 3 to 5 AF cells were plated in PLO / LN (poly-L-ornithine or poly-L-lysine) coated tissue culture plates and infected with Oct4 or Sox2 lentivirus particles at MOI of 10-100 in the presence of 8 μg / ml polybrene for 6 hours. 24-48 hours after infection, the efficiency of lentiviral infection was determined under fluorescent microscope by GFP expression. Typically the percentage of GFP positive cells can reach higher than 80%.

[0126]Transduced cells were plated to assess growth. Numerous colonies were present only 7 or 9 days post-transduction. The OCT4 transduced colonies in particular were able to expand and continue to grow over 10 passages. The inventors found that addition of small chemical compounds such as TGF-βreceptor I inhibitor II (10 uM) a...

example 2

OCT4 or SOX2 Overexpression via Lentiviral Transduction can Reprogram AF Cells to Neural Precursor Cells

[0127]After OCT4- or SOX2-induced dedifferentiation, cells were transferred to ReNcell medium plus bFGF and EGF. On Day 4 in ReNcell medium, OCT4 and SOX2-induced AF cells formed cell clusters in culture (FIG. 1C; FIG. 7, “AF”) and these clusters could be dissociated into single cells for monolayer culture. The induced cells resembled neural stem cells in morphology (FIG. 1 D; FIG. 6“SOX2”), including development of long, thin processes which are drastically different from the WT or GFP-only control cells (FIGS. 1A-B; FIG. 6“GFP” and “WT”).

[0128]Neurosphere formation is a feature of NSC growth in vitro. Not surprisingly, the induced NSC from AF cells could form typical neurospheres in low attachment dish (FIG. 2A; FIG. 7“AF”).

[0129]Next, the inventors used antibodies to Nestin and Mushashi-1, which are neural stem cell markers, to detect the expression of NSC-specific markers in t...

example 3

OCT4 Overexpression via Lentiviral Transduction can Reprogram AF Cells to Endothelial Stem Cells

[0133]After viral transduction, cells were transferred to human ES (embryonic stem cell) medium (supplemented with 20% Knockout Serum replacement, 2 mM glutamine, 0.1 mM non-essential amino acids, 10 ng / ml bFGF, 100 μM β-Mercaptoethanol) or endothelial cell medium, such as EGM2 (Lonza, Inc.). Numerous colonies appeared on day 9 that were not seen in the AF cells transduced with control lentiviruses expressing GFP. More than 90% of colonies were FLK1 positive by day 11.

[0134]In order to increase the colony forming efficiency, the inventors tried different culture media and found that the addition of TGF-beta receptor1 inhibitor II, 616452 at 10 μM or 8-Br-cAMP at 0.1 mM in the culture media accelerated and increased the efficiency of colony formation by approximately 15%.

[0135]616452 is 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine with structure:

[0136]8-Br-cAMP, also know...

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Abstract

Presented herein are methods of generating an induced stem cell (iSC) from a somatic cell, by contacting the somatic cell with an induction factor that reprograms the somatic cell to generate an iSC. The induction factor can be a genetic construct or a fusion protein. Where the induction factor is a genetic construct, the construct bears one or more nucleotide sequences encoding one or more reprogramming elements selected from OCT4, SOX2, NANOG, and a Notch pathway molecule, or an active fragment or derivative thereof. The genetic construct can have a lentiviral or episomal vector backbone. The induction factor can also be a fusion protein, with the reprogramming element being a protein selected from OCT4, SOX2, NANOG, or a Notch pathway molecule, or an active fragment or derivative thereof. The fusion protein can be TAT protein or an active fragment or derivative thereof.

Description

BACKGROUND OF THE DISCLOSURE[0001]Stem cells are undifferentiated cells that have extensive proliferation potential, can differentiate into several cell lineages, and repopulate tissues upon transplantation. Stem cells can give rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable daughter cells. The quintessential stem cell is the embryonic stem cell, as it has unlimited self-renewal and pluripotent differentiation potential (Orkin, Int. J. Dev. Biol. 42:927-34, 1998; Reubinoff et al., Nat Biotech. 18:399404, 2000; Shamblott et al., Proc. Natl. Acad. Sci. U.S.A. 95:13726-31, 1998; Thomson et al., Science 282:114-7, 1998; Thomson et al., Proc. Natl. Acad. Sci. USA. 92:7844-8, 1995; Williams et al., Nature 336:684-7, 1988). Embryonic stem (ES) cells have been extensively studied for use in providing sources of new tissue. However, in addition to the ethical and supply issues surround...

Claims

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Application Information

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IPC IPC(8): C12N5/074A61K35/12C12N5/0797
CPCC12N5/0696A61K35/12C12N5/0623C12N2501/01C12N2501/15C12N2501/602C12N2501/603C12N2506/025C12N2506/1353C12N2506/1392C12N2510/00A61K35/545
Inventor MA, YUPOLIAO, WENBIN
Owner THE RES FOUND OF STATE UNIV OF NEW YORK
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